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424

12.1 Introduction 424

12.2 Basic Principles of Sedimentation 425

12.3 Types, Care and Safety Aspects of Centrifuges 430 12.4 Preparative Centrifugation 438

12.5 Analytical Ultracentrifugation 446

12.6 Suggestions for Further Reading 452

12.1 INTRODUCTION

Biological centrifugation is a process that uses centrifugal force to separate and purify

mixtures of biological particles in a liquid medium. The smaller the particles, the higher the g-forces (see next section ) required for the separation. It is a key technique for iso- lating and analysing cells, subcellular fractions, supramolecular complexes and, with higher g-force instruments or ‘ultra'-centrifuges (up to 60-000 revolutions per min- ute corresponding to ~ 200 000×g) isolated macromolecules such as proteins or nucleic acids. Such high-speed devices require a vacuum to avoid overheating of samples. The development of the fi rst analytical ultracentrifuge - with a specially designed optical system for monitoring and recording the sedimentation process - by Svedberg in the late 1920s and the technical refi nement of the preparative centrifugation technique by Claude and colleagues in the 1940s positioned centrifugation technology at the centre of biological and biomedical research for many decades. Today, centrifugation techniques represent a critical tool for modern biochemistry and are employed in almost all inva- sive subcellular studies. While analytical ultracentrifugation is mainly concerned with the study of purifi ed macromolecules or isolated supramolecular assemblies, prepara- tive centrifugation methodology is devoted to the actual separation of tissues, cells, subcellular structures, membrane vesicles and other particles of biochemical interest. Most undergraduate students will be exposed to preparative centrifugation protocols during practical classes and might also experience a demonstration of analytical cen- trifugation techniques. This chapter is accordingly divided into a short introduction into the theoretical background of sedimentation, an overview of practical aspects of using centrifuges in the biochemical laboratory, an outline of preparative centrifugation and a description of the usefulness of ultracentrifugation techniques in the biochemical

characterisation of macromolecules. To aid in the understanding of the basic principles KAY OHLENDIECK AND STEPHEN E. HARDING

Centrifugation and

Ultracentrifugation 12 Book 1.indb 42409/12/17 4:24 PM

42512.2 Basic Principles of Sedimentation

of centrifugation, the general designs of various rotors and separation processes are diagrammatically represented. Often, the learning process of undergraduate students is hampered by the lack of a proper linkage between theoretical knowledge and practical applications. To overcome this problem, the description of preparative centrifugation techniques is accompanied by an explanatory μ ow chart and the detailed discussion of the subcellular fractionation protocol for a specifi c tissue preparation. Taking the isolation of fractions from skeletal muscle homogenates as an example, the ratio- nale behind individual preparative steps is explained. Since affi nity isolation methods not only represent an extremely powerful tool in purifying biomolecules (see Chapter

5 ), but can also be utilised to separate intact organelles and membrane vesicles by

centrifugation, lectin affi nity agglutination of highly purifi ed plasmalemmal vesicles from skeletal muscle is described. Traditionally, marker enzyme activities are used to determine the overall yield and enrichment of particular structures within subcellular fractions following centrifugation. As an example, the distribution of key enzyme activities in mitochondrial subfractions from liver is given. However, most modern fractionation procedures are evaluated by more convenient methods, such as protein gel analysis in conjunction with immunoblot analysis ( Chapter 7 ). Miniature gel and blotting equipment can produce highly reliable results within a few hours, making it an ideal analytical tool for high-throughput testing. Since electrophoretic techniques are introduced in Chapter 6 and are used routinely in biochemical laboratories, the protein gel analysis of the distribution of typical marker proteins in affi nity-isolated plasmalemma fractions is graphically represented and discussed. Although monomeric peptides and proteins are capable of performing complex bio- chemical reactions, many physiologically important elements do not exist in isolation under native conditions. Therefore, if one considers individual proteins as the basic units of the proteome (see Chapter 21 ), protein complexes actually form the functional units of cell biology. This gives investigations into the supramolecular structure of protein complexes a central place in biochemical research. To illustrate this point, the sedimentation analysis of a high-molecular-mass membrane assembly, the dystrophin-glycoprotein complex of skeletal muscle, is shown and the use of sucrose gradient centrifugation explained. Analytical ultracentrifugation - which unlike other analytical separation tech- niques does not require a separation medium, i.e. it is ‘matrix-free' - has become a preferred or ‘gold standard' technique for establishing the purity or homogeneity and state of aggregation of macromolecular or nanoparticle solutions, and to illustrate this point, we show how the purity of preparations of monoclonal antibodies can be rou- tinely analysed with the modern ultracentrifuge, and how the inclusion of a density gradient, when appropriate, can enhance the resolution of the method even further.

12.2 BASIC PRINCIPLES OF SEDIMENTATION

From everyday experience, the effect of sedimentation due to the inμ uence of the Earth's gravitational fi eld ( G = g = 9.81 m s 2 ) versus the increased rate of sedimen- tation in a centrifugal fi eld ( G > 9.81 m s 2 ) is apparent. To give a simple but illus- trative example, crude sand particles added to a bucket of water travel slowly to the bottom of the bucket by gravitation, but sediment much faster when the bucket is swung around in a circle. Similarly, biological structures exhibit a drastic increase

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Centrifugation and Ultracentrifugation426

in sedimentation when they undergo acceleration in a centrifugal eld . The relative centrifugal fi eld is usually expressed as a multiple of the acceleration due to gravity. Below is a short description of equations used in practical centrifugation classes. When designing a centrifugation protocol, it is important to keep in mind that: the more dense a biological structure is, the faster it sediments in a centrifugal fi eld the more massive a biological particle is, the faster it moves in a centrifugal fi eld the denser the biological buffer system is, the slower the particle will move in a cen- trifugal fi eld the greater the frictional coef cient is, the slower a particle will move the greater the centrifugal force is, the faster the particle sediments the sedimentation rate of a given particle will be zero when the density of the particle and the surrounding medium are equal. Biological particles moving through a viscous medium experience a frictional drag , whereby the frictional force acts in the opposite direction to sedimentation and equals the velocity of the particle multiplied by the frictional coeffi cient. The frictional coef- fi cient depends on the size and shape of the biological particle. As the sample moves towards the bottom of a centrifuge tube in swing-out or fi xed-angle rotors (see Section

12.3.2 ), its velocity will increase due to the increase in radial distance. At the same time,

the particles also encounter a frictional drag that is proportional to their velocity. The frictional force of a particle moving through a viscous μ uid is the product of its veloc- ity and its frictional coeffi cient, and acts in the opposite direction to sedimentation. When the conditions for the centrifugal separation of a biological particle are described, a detailed listing of rotor speed and radial dimensions of centrifugation has to be provided. Essentially, the rate of sedimentation, v , is dependent upon the applied centrifugal eld G (measured in cm s -2 ). G is determined by the radial distance, r , of the particle from the axis of rotation (in cm) and the square of the angular velocity ,

π, of the rotor (in radians per second):

G = π

2 × r (Eq 12.1) The average angular velocity of a rigid body that rotates around a fi xed axis is defi ned as the ratio of the angular displacement in a given time interval. One radian, usually abbreviated as 1-rad, represents the angle subtended at the centre of a circle by an arc with length equal to the radius of the circle. Since 360° equals 2 radians (or rad), one revolution of the rotor can be expressed as 2 rad. Accordingly, the angular velocity of the rotor, given in rad s -1 . Note that rad is treated as a scalar and is related to the rotor speed in revolutions per minute (rpm = 1 min -1 ) by π= 2π rad × rpm (Eq 12.2) and therefore the centrifugal fi eld can be expressed as:

G = 4π

2 rad 2

× rpm

2 × r (Eq 12.3) where the variable rpm is the rotor speed (measured in revolutions per minute, i.e. the non- italicised ‘rpm' denotes the unit) and r is the radial distance from the centre of rotation. Note that 60 revolutions per minute is the same speed as one revolution per second, i.e. rpm = 60 min -1 = 1 s -1

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42712.2 Basic Principles of Sedimentation

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